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Anal. Chem. 1987, 59,2428-2432
that probabilities of highest ranking can be estimated for all two-dimensional systems considered, according to the size of the subset, and using either the P R F or the IDF as a ranking criterion; that r values are substantially influenced by the size of the subsets considered but that in most cases are not substantially influenced by whether the P R F or IDF are used as ranking criteria; that the identity of either the very highest ranked or very lowest ranked solvent systems for separating subsets of a given size is not affected by which function is used as a ranking criterion although the ranking of intermediate solvent systems is influenced by which function is used. A similar study is currently being performed with the same two-dimensional systems but with a different mixture of steroids, as well as with other classes of compounds. This statistical approach to the selection of separation systems should be applicable, with suitable modification, to solvent selection in one-dimensional TLC, to stationary phase selection in gas chromatography, and to solvent selection in column liquid chromatography. It should apply to any class of compounds for which there is an empirical relationship
between solute capacity factor and solvent composition.
LITERATURE CITED (1) Gonnord, M.-F.; Levi, F.; Guiochon, G. J . Chromatogr. 1983, 264. 1-6. (2) Johnson, E. K.; Nurok, D, d . Chromatogr. 1984, 302, 135-147. (3) Steinbrunner, J. E.; Johnson, E. K.: Habibi-Goudarzi, S.; Nurok. D. I n Planar Chromatography; Kaiser, R. E., Ed.; Heuthig Verlag: Heidelberg, 1986; Vol. 1: p 239. (4) Snyder, L. R.; Kirkland, J. J. Introduction to Modern Liquid Chromatography; Wiley: New York, 1979. (5) Poole, C. F.; Schuette, S. A. Contemporary Practice of Chromafogra phy; Elsevier: Amsterdam, 1984. (6) Morgan, S. L.; Deming, S. N. J . Chromatogr. 1975, 772, 267-285. (7) Kaiser, R. E. Gas Chromatographie; Geest 8 Portig: Leipzig, 1960; p 33. (8) Hollander, M.; Wolfe. D. A. Nonparametric Stafistical Methods ; Wiley: New York. 1973. ~
RECEIVED for review December 9, 1986. Resubmitted June 2, 1987. Accepted June 2, 1987. This work was supported by a grant from the Dow Analytical Laboratories University Support Program. The TLC plates were a gift from Whatman Chemical Separations, Inc.
Preparation and Evaluation of Slurry-Packed Capillary Columns for Normal-Phase Liquid Chromatography Franca Andreolini, Claudio Borra, and Milos Novotny* Department of Chemistry, Indiana University, Bloomington, Indiana 47405
Effective procedures have been developed to pack fused slllca (Capillary) columns with 5-pm particles of slllca adsorbent and other polar materials (diol-, cyano-, and amlnobonded phases). Mlcrocdumns exhlbltlng overall efflclencles between 70 000 and 90 000 theoretical plates were prepared. The reduced plate heights, separatlon Impedance, and sample capaclty of such columns were further evaluated.
The sample complexity often encountered with biological, environmental, and technologically important nonvolatile mixtures demands high-efficiency separation techniques. Consequently, the interest in microcolumns of capillary dimensions for use in liquid chromatography (LC) has grown rapidly during the last several years. The column technology of highly efficient and stable slurry-packed capillary columns is a subject that deserves primary attention. While several reports have now been published concerning the preparation and evaluation (1-5) of reversed-phase, slurry-packed capillary columns, virtually no investigations have been performed on the packing techniques and quantitative aspects of normal-phase columns with similar dimensions. Straight-phase separations encompass adsorption chromatography on silica gel and alumina (although the latter is rarely used) and partition chromatography on polar chemically bonded phases. Some desirable features of normal-phase chromatography are (a) the ability to obtain a class separation selectively, (b) the capability of resolving certain isomers, which are of great significance in the analysis of natural products and in pharmaceutical chemistry, (c) the ability to separate hydrophilic species that cannot be easily retained 0003-2700/87/0359-2428501.50/0
in the reversed-phase systems, (d) the ability to differentiate solutes based on the differences in hydrophilic rather than hydrophobic structure, (e) compatibility of organic phases with molecules that have either low stability or aggregation problems in aqueous phases, and (0 the availability of a wide range of stationary-phase selectivities. All these features make normal-phase liquid chromatography with high-efficiency capillary columns quite attractive. The use of capillary columns, characterized by flow rates of 1-3 ML/min, allows the routine use of expensive, ultrapure mobile phases, which are demanded for a reliable practice of adsorption chromatography. In addition, “exotic” mobile phases (e.g. deuteriated or chiral solvents) could also be used in this type of chromatography. And, moreover, the lower polarity, higher volatility, lower gas expansion volumes and wetting characteristics of organic mobile phases, combired with a low flow rate, confer an advantage in coupling straight-phase capillary columns to mass spectrometers, infrared instruments, and the detection techniques that require solvent elimination. This communication describes the packing procedures developed for slurry-packed capillary columns, using silica gel and polar chemically bonded stationary phases of the diol, cyano, and amino types. Markedly different packing procedures for each material had to be employed in order to obtain optimum results. Performance of the prepared columns has been evaluated through the reduced plate height vs. reduced velocity plots and the separation impedance values. In addition, reproducibility of the packing procedures and the column loading capacities has been assessed.
EXPERIMENTAL SECTION Column Packing. After a porous PTFE frit was fixed at the
column end ( 6 ) ,fused silica capillaries (Polymicro Technologies, 0 1987 American
Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 19, OCTOBER 1, 1987
Table 11. Column Test Conditions
Table I. Optimum Packing Conditions packing material slurry ratio (v/w) solvent acetonitrile/methanol methanol / water/ diethylamine procedure
diol 511
cyano amino 4/1
4/1
50/50 75/25
silica 41 1
capacity ratio diol cyano amino silica 9713" 9515" 97/30 9713" 1.9 pL/min 1.6 pL/min 1.2 pL/min 1.7 pL/min
75/25 90/ 10/0.1
fastn
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slowb fast"
slowb
OStarted at 50 atm and increased by 50 atm, every 5 s, up to 450 atm and then kept under pressure for 30 min. bStarted at 10 atm and increased by 10 atm every 15 min. When the column was fully packed, the pressure was set at 350 atm and remained there for 30 min. During the packing procedure, t h e column was placed in the ultrasonic bath. Phoenix, AZ), 1 m X 250 pm i.d. and 350 pm o.d., were slurrypacked by using a Model LC-5A pump (Shimadzu, Kyoto, Japan). The slurry reservoir, having a totalvolume of ca. 200 pL, consisted of a short piece of stainless-steel tubing, 1.6 mm id., directly connected to the fused silica capillary through a reducing union. The Spherex diol phase was purchased from Phenomenex (Rancho Palm Verdes, CA), while the other phases, Spherisorb S,W, CN, and NH2,were obtained from Phase Sep. (Norwalk, CT). All four packing materials had a nominal particle size of 5 pm. Table I shows the packing procedures that gave the best results for each of the packings tested. When the slow packing technique (Table I) was used, the column and the slurry reservoir were totally immersed in a 110 X 5 cm i.d. glass cylinder filled with water. The cylinder base was set in an ultrasonic bath to prevent particle aggregation. After the pump was stopped, the pressure was allowed to decrease slowly. Once the pressure returned to zero, the column was equilibrated with the mobile phase used to evaluate the column performance. In the case of the amino columns, an intermediate washing step with 2-propanol was applied to remove water and diethylamine used during the packing procedure. Column Evaluation. The chromatographic system utilized for the column evaluation consisted of a Model pLC-500 syringe pump (ISCO, Lincoln, NE) operated in the constant-flow mode, a UV absorbance detector (UVIDEC-100-V,Jasco, Tokyo, Japan) with an in-house modified 10-nL cell employed to detect the solutes a t 254 nm, and a four-port internal loop (electrically activated) injection valve (Model ECI4W, Valco Instruments, Houston, TX) with a 0.2-pL rotor. T o obtain a flow cell compatible with the stringent volumetric requirements of capillary columns, a short piece of 50 pm i.d. fused silica capillary, with the polyamide coating removed where the light beam crosses it, was used to replace the original detector cell. Since the mobile-phase laminar profile causes the sample to be delayed along the loop wall, a sample volume totally injected into the column would have an exponential decay profile. To avoid this undesirable effect, the moving-injection technique ( 7 ) was employed. The injection time and, consequently, the injected sample amount were controlled by an IBM personal computer connected to the electrical actuator. The same computer, employing in-house developed programs, acquired and calculated the peak moment data. Table I1 details a test mixture, mobile phase, and flow rate that have been used to evaluate each packed column. The solvent of all the test mixtures was hexane; its peak was used to approximate the dead time. The compounds present in each mixture were chosen so as to cover a range of different chemical characteristics and capacity factors. The numbers of theoretical plates (N) and, thus, the reduced plate-height values (h) were obtained through the moment analysis. These values, combined with the time necessary to elute an unretained peak ( t o ) ,the pressure drop required t o achieve and the solvent viscosity (q), were used to the separation (AP), calculate the values of separation impedance (E) (8).
As suggested by Bristow and Knox (8),E is an index of column
anthracene nitrobenzene 2,6-dimethylaniline acetophenone dimethyl phthalate triphenylmethanol phenol o-nitroacetophenone
0.11
0.31
0.15
0.47
0.76
ntb
nt
nt 0.56
nt
nt
nt
0.60
1.40
1.50
0.77
1.11
nt
nt
1.12
nt
nt
1.62 nt
nt nt
1.57 nt
2.52
0.14 0.47
nt
aMobile uhase: uentanelethanol. brit = not tested. global performance, which is dimensionless. The lower its value, the better the column performance. Values as low as 2000 can be obtained for packed columns, while the column resistance parameter (p) for conventional (4.6 mm i.d.), 1 mm i.d., and slurry-packed capillary columns normally falls in the range of 500-1000 (5, 9). The reduced velocities ( u = Ud,/D,, where U = mobile-phase velocity, d , = particle diameter, and D, = diffusion coefficient of a solute in the mobile phase) were determined by using D, values calculated through the empirical Wilke-Chang equation (10) and the mobile-phase velocities.
RESULTS AND DISCUSSION The optimum flow rates for chromatographic efficiency of each packing material were determined. Comparison of the van Deemter curves obtained with all column types is shown in Figure 1. Although selected columns are demonstrated here with several model solutes, the remaining successfully prepared microcolumns yielded results similar t o those illustrated in this figure (see below). At the mobile-phase velocities close to optimum (v = l),the lowest h values, hmin, for the diol phase and the cyano packing were in the range of 2.7-2.9 and 2.2-2.5, respectively. The best efficiencies obtained for the amino phase were somewhat lower, resulting in hminfrom 2.9 t o 3.1 and even 3.5 for triphenylmethanol. From the four stationary phases studied here, the amino columns were the hardest t o optimize as far as the packing procedure is concerned. With silica gel as the column material, a broad range of hmh values was found for different test compounds (Figure 1). While the result for acetophenone, h- ca. 2.6, was comparable to those achieved with the other packings, the values for anthracene, nitrobenzene, and dimethyl phthalate were considerably higher (hminaround 3.5). Phenol, a polar protic compound, fared even worse, with h- of 4.3. The differences between the solutes of different polarities in yielding variable peak widths and symmetries are not unexpected, due t o the adsorption basis of retention. Results such as these can frequently be improved through the use of a suitable mobile-phase modifier. The values of u at which the best efficiencies were recorded were lower than those obtained previously with slurry-packed, reversed-phase capillary columns (5). Interestingly, the hmin for silica and amino phases obtained here were slightly lower than those observed with (1m m i.d.) microbore columns (11) and considerably lower than those usually reported for conventional columns (8). Such a trend, the lowering of the optimum flow rate as the column internal diameter decreases, has been reported also for reversed-phase liquid chromatography (12). These results appear t o be a consequence of a
2430
ANALYTICAL CHEMISTRY, VOL. 59, NO. 19, OCTOBER 1, 1987 cyano 5.5
'1
4.5
h
I
4:
+
+ A A 0
A
+
o+
04 D
0
d'
0
0
oo
Q
5.5
-
5.5
x X X
S-
5 4.5
4
-
I
4.5
h
P
-
X
X
x
O
X
x
+
4
D
4-
-
0
++
4
D e * o
3.5
4
+ D
n+ e n
00
A ¶.
A
1
3-
2.5
1
2.5
2
j 0.4
0.6
0.8
1
1.2
2
4 1.4
1.6
*
1.1
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1 0.4
2
0.6
d
0.8
/ 1
1.2
1.4
1.6
1.8
2
V
V
2,6-dimethylaniline (m), Flgure 1. Plots of h vs. v for diol, cyano, amino, and silica gel packings. Solutes: anthracene (+), nitrobenzene (O), acetophenone (A),dimethyl phthalate (0),triphenylmethanol (V),phenol (X), o -nitroacetophenone (A). lowered axial dispersion encountered with the columns of smaller diameters. Following the packing procedures described in this report, it is quite easy to prepare 1-m long columns, yielding 70 000-90 000 theoretical plates, for both silica- and polarbonded materials. Such chromatographic efficiencies are roughly comparable to those obtained with the reversed-phase slurry-packed capillary columns ( 5 ) . I t must to be pointed out, however, that, just as is known for conventional columns, markedly different packing procedures are required for packing materials with different surface chemistry. We strongly suspect that even materials of similar types, originated from different commercial sources, may yield somewhat different results. Consequently, the packing procedures described here should be retested, and possibly modified, if packing materials from different sources are employed. The slow packing procedure, utilized optimally for the silica and cyano packings, is quite different from what has been published and previously employed with conventional (13-19, microbore (II), or slurry-packed capillary columns (2-5). Takeuchi et al. ( I ) used low pressures to pack their microcolumns. However, these authors were able to efficiently pack only short columns (100-1000-fold higher in concentration than formaldehyde to be significant ( I ) . None of these interferents are very likely to be significant when domestic or occupational indoor air is sampled to determine formaldehyde levels. A major advantage that the chromotropic acid method has over the modified pararosaniline method is that it can be used to determine formaldehyde that has been collected in aqueous bisulfite solution. This medium is reported to be preferable to distilled or deionized organic-free water (6). I t is possible however that the greater sensitivities noted could be due to the fact that dilute aqueous formaldehyde solutions have been shown to be stabilized by the presence of sodium bisulfite (7). Experiments in which side-by-side replicate determinations of indoor air formaldehyde were conducted with midget glass impingers containing only deionized organic-free water and impingers containing water of similar quality but also 1% sodium bisulfite showed no significant differences when analyzed by the chromotropic acid method. The samples obtained in these experiments either were analyzed immediately after sampling or were refrigerated within 1 t o 2 h after sampling and were analyzed 24 to 48 h after sampling (8). Other studies have shown, however, that concentrations of dilute formaldehyde in aqueous solutions not stabilized with bisulfite (7), methanol (7), or bacteriocides such as mercury(I1) chloride, tin(I1) chloride, or sodium pentachlorophenate (9) rapidly decrease with time even when refrigerated (7). Since bisulfite is a known interferant ( 1 , 5 )in the modified pararosaniline method, formaldehyde solutions stabilized in
this way cannot be analyzed by the method. The use of dilute pararosaniline solution as a trapping medium for formaldehyde was investigated during the development of an assay kit for the measurement of formaldehyde in indoor air (IO). We herein report on our observations on the storage stability of dilute formaldehyde solutions containing pararosaniline reagent.
EXPERIMENTAL SECTION Apparatus. Spectrophotometric determinations were made with a Perkin-Elmer Hitachi-Coleman 139 grating spectrophotometer. Matched optical glass 10-mm path length cuvettes were used. Oven-dried clean glass 25-mL culture tubes capped with Teflon-lined screw caps were used throughout to store the formaldehyde solutions and blanks. Solutions (20 mL) of known formaldehyde concentrations and the correspondingblanks were pipetted into each of the clean culture tubes, using disposable sterile pipets. The culture tubes were capped and sealed on the outside with strips of Parafilm. Reagents. Pararosaniline (free base) (Cl9HI9N30)and pararosaniline hydrochloride (C,gH,8N3Cl)were purchased from Sigma Chemical Co. Stock pararosanilinereagent (PRA) was purchased from CEA Instruments, Inc., and was acidified by the addition of sufficient concentrated hydrochloric acid to bring its [H+]to 1.68 M as described in ref 4. Stock methanol-free formaldehyde solutions were prepared and standardized by the methods described by Miksch et al. ( I ) . Their pararosaniline procedure was employed with the modifications described in ref 4. Three 1.0-L batches each of solutions corresponding to formaldehyde concentrations of 0.046,0.092, and 0.184 pg/mL were prepared from the stock solution which had previously been standardized, by dilution of the appropriate amounts of the stock solution with organic-freedeionized water. Three 1.0-L batches of solutions corresponding to the same formaldehyde concentrations as above were prepared in the same manner but in addition, 90 mL of the pH-adjusted PRA was added to each solution. Thus for example, for the solution containing 0.046 pg/mL formaldehyde in organic-free deionized water, 4.6 mL of a stock solution of formaldehyde whose concentration was determined to be 10.0 pg/mL was added to a 1.0-L volumetric flask and the volume made up to 1.0 L with organic-freedeionized water. The corresponding solutions which contained 10% PRA were prepared
0003-2700/87/0359-2432$01.50/0 'C 1987 American Chemical Society
